Since uncultivable microorganisms comprise a large percentage of the microbiome, and are likely to play a major role in the ecology at all sites within the body, it is critical to develop new approaches to obtain samples of these microorganisms for genomic analysis. In this proposal we focus on one anatomic site, the mouth, and propose to develop a technology to extract single bacterial cells from saliva. To attain these goals, we have formed a collaboration between Sandia (with expertise in integrated microfluidic technology for biological analysis), NYU College of Dentistry (with expertise in oral microbiomics and oral-based diagnostics), and the Joint Genome Institute (with expertise in microbial ecology and sequencing). The technological approach is to build an integrated microfluidic cell processor that will identify, select, and isolate into discrete microdroplets single bacteria from a mixture of oral bacteria from human saliva. The microfluidic processor will have multiple modules to 1) perform fluorescence in situ hybridization on a mixture of bacteria, 2) sort single cells using fluorescence activated photonic-force deflection, and 3) encapsulate sorted cells in microdroplets before depositing them on an array. The input to the device will be bacterial cells from saliva and the output will be arrayed droplets containing no more than one bacterium. We will first characterize and validate this processor using a mixture of pure bacterial cultures. Subsequently we will take salivary samples, deplete them of abundant bacterial species, and isolate individual cells, using specific 16S probes. Metagenomic analysis on the entire population of bacteria in saliva will be used to identify new bacterial sequences. With new sequence information, we will design 16S probes to isolate previously uncharacterized organisms for genomic testing. Isolated cells will be characterized as cultivable or non-cultivable, and known (sequenced) or unknown. Ultimately this technique will be used to extract sequence-quality genomic DNA from individual microorganism and can be used as a diagnostic to identify bacterial signatures obtained from healthy versus diseases subjects. PUBLIC HEALTH RELEVANCE: We know little about bacteria found in our bodies that can not be grown in the laboratory, but which may nevertheless cause disease. Our goal is to develop lab-on-a-chip technologies for isolating organisms from clinical samples and processing them, one at a time, for further genomic analysis.